In the period covered by this review, we have stayed focussed on understanding the cellular and molecular biology of genes associated with Parkinson?s disease or parkinsonism. The number of genes unambiguously associated with Parkinson?s disease parkinsonism has increased to five, including two poorly characterized kinases (PINK1 and LRRK2). Mutations in the gene for PINK1, a serine/threonine kinase, are associated with autosomal recessive parkinsonism. In the last year we have confirmed preliminary reports that the protein is localized to mitochondria and have shown that there is a leader peptide of approximately 80 amino acids is cleaved from the C-terminus of the protein after import into the organelle. We also showed that recombinant proteins generated without a leader peptide and expressed in E coli have detectable kinase activity, in line with predictions based on similarity to other kinases. This allowed us to measure kinase activity of mutant PINK1, either recessive mutations associated with human disease or artificial mutants designed to test which residues are critical for kinase activity. Surprisingly, one mutation associated with parkinsonism (G309D) retained partial activity in this assay. However, another mutation (L347P) had very little activity but was also unstable when expressed in this heterologous system. This was reminiscent of mutations in DJ-1, which we have previously shown to be unstable in cell culture models (see below). We were able to show that L347P PINK1 is also unstable in mammalian cells, suggesting that a lack of protein stability leads to disease in these cases. A frustration has been that we were unable to test activity against authentic substrates, as we used the convenient but artificial autophosphorylation assay to address activity. However, recent unpublished results suggest that PINK1 has at least in vitro activity towards several other kinases, a result that we will attempt to confirm in more physiological settings in the next year. This work on PINK1 relates to previous work on stability of DJ-1 that we have also extended recently to show that protein stability contributes to the loss of function of two mutations (L166P and M26I) but not several other variants that are probably causal mutations. We have shown that the immunolocalization of DJ-1 can be mitochondrial, like PINK1, but that mutations have complex effects on this phenotype. Several mutations increase the association with mitochondria in our hands, but only under oxidative conditions. We have also mapped epitopes for commercial and novel monoclonal antibodies to DJ-1. This is important to us as our results for protein localization depend on understanding epitope availability, which might affect how much DJ-1 appears to be mitochondrial. A major novel line of investigation is to define the molecular biology of dardarin (gene name LRRK2), a large multifunctional kinase associated with autosomal dominant parkinsonism. Preliminary experiments show that dardarin is a >250 kDa cytosolic protein. Unlike PINK1 or DJ-1, the pathology of dardarin cases is known. There is variability, but cases can have either alpha-synuclein or tau positive inclusion bodies. We have worked on alpha-synuclein for several years and an obvious set of experiments, underway in the laboratory, is to define the relationship(s) between dardarin and synuclein.